Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: Human TMEM30a partially reconstitutes phospholipid import in ⊗Lem3 S. cerevisiae

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques:

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) ΔLem3 S. cerevisiae transformed with empty vector or two isolates transformed with human TMEM30a were grown on glucose or galactose to induce TMEM30a expression. NBD-phosphatidylcholine uptake was determined by flow cytometry. (B) Concentration dependent effect of Edelfosine on colony growth of serially diluted wild-type S. cerevisiae or ΔLem3 transformed with empty vector or two ΔLem3 isolates transformed with human TMEM30a.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transformation Assay, Plasmid Preparation, Expressing, Flow Cytometry, Concentration Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake determined by flow cytometry for wild-type S. cerevisiae transformed with empty vector or ΔLem3 transformed with Lem3, TMEM30a or a chimera (Table 1) of Lem3 and TMEM30a. (B) Quantitation (n=3) of NBD-phosphatidylcholine uptake by ΔLem3 transformed with Lem3-TMEM30a (LT; see Table 1 for sequence), TMEM30a-Lem3 (TL), or TMEM30a-Lem3-TMEM30a (TLT) chimeras. Western blot (top) for V5 antigen contained in sequences encoding TMEM30a and its chimeras isolated from protein extracts of S. cerevisiae grown in galactose to induce insert expression or non-inducing glucose. (C) Concentration dependent effect of Edelfosine on colony formation on glucose or galactose plates for wild-type S. cerevisiae or ΔLem3 transformed with galactose induced human, yeast or chimeric constructs. (D) Effect of Edelfosine on ΔLem3 viability after introduction of human TMEM30a, yeast Lem3p, or chimeras formed from them. Cell number (OD600) in liquid culture of wildtype or ΔLem3 transformed with the stated vectors at defined concentrations (left) or 12.5 μg/ml (right).

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Flow Cytometry, Transformation Assay, Plasmid Preparation, Quantitation Assay, Sequencing, Western Blot, Isolation, Expressing, Concentration Assay, Construct

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) CHO cells stably transfected with TMEM30a-GFP and then stained with CellMask™ Orange Plasma Membrane to mark the plasma membrane (top) then imaged by confocal microscopy. Co-expression of the appropriate orange fluorescent protein Organelle Light defined endoplasmic reticulum (row 2), or Golgi (row 3). TMEM30a-GFP expressing CHO cells were labeled with MitoTracker Red to identify polarized mitochondria (bottom). (B) Western blot for GFP or plasma membrane Na/K ATPase in density gradient fractions from HepG2 cells stably expressing TMEM30a-GFP. (C) Fluorescent intensity of TMEM30a-Jurkat cells during flow cytometry after 10 min incubation in the presence of NBD-phosphatidylcholine (1 μM) alone or additionally with 5 μM Az-LPAF or Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Stable Transfection, Transfection, Staining, Confocal Microscopy, Expressing, Labeling, Western Blot, Flow Cytometry, Incubation

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) NBD-phosphatidylcholine uptake by CHO cells transfected with empty vector or a TMEM30a vector assessed by confocal microscopy (40X). Inset, 60X. (B) Uptake of [3H]PAF by CHO cells expressing TMEM30a containing a GFP or Lumio tag (n=3). (C) Phosphatidylserine surface expression is not reduced in TMEM30a transfected CHO cells. Surface phosphatidylserine was detected (n=3) by flow cytometry with annexin V conjugated with Alexa647 as described in “Methods.”

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Transfection, Plasmid Preparation, Confocal Microscopy, Expressing, Flow Cytometry

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Quantitative PCR for TMEM30a mRNA after transfection by empty vector or one containing TMEM30a shRNA (n=3). (B) Jurkat viability to Edelfosine exposure after transfection with an empty vector or TMEM30a shRNA (n=3). (C) Jurkat cell uptake of fluorescent NBD-phosphatidylcholine (upper) or NBD-phosphatidylethanolamine (lower) by cells expressing TMEM30a shRNA or its vector (n=3). (D) Quantitation of NBD-phosphatidylcholine accumulation by Jurkat cells expressing TMEM30a shRNA or empty vector (n=3). (E) Uptake of [3H]PAF by Jurkat cells is reduced by TMEM30a shRNA knockdown (n=4). All quantitative measures used triplicate determinations in each experiment.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Quantitation Assay

Journal:

Article Title: Human TMEM30a Promotes Uptake of Anti-tumor and Bioactive Choline Phospholipids into Mammalian Cells 1

doi: 10.4049/jimmunol.1002710

Figure Lengend Snippet: (A) Flow cytometric analysis of JC-1 green fluorescence (FL1, x axis) and orange/red fluorescence (FL2, y axis) in the presence of the stated azelaoyl lysoPAF concentration in vector and TMEM30a shRNA transfected Jurkat cells. The cationic dye JC1 in functional, polarized mitochondria is aggregated and fluoresces red/orange, while monomeric dye free in the cytoplasm fluoresces green. (B) Flow cytometric analysis of JC-1 fluorescence in the stated concentration of Edelfosine.

Article Snippet: Human TMEM30a cDNA was purchased from Origene Technologies (Rockville, MD), and shRNA plasmids against human TMEM30a were from SuperArray Bioscience (Frederick, MD).

Techniques: Fluorescence, Concentration Assay, Plasmid Preparation, shRNA, Transfection, Functional Assay

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet: mTORC2 inhibition prevents proliferation and self-renewal in GSCs (A) IB analysis of CD97, p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) infected with shCtrl or shCD97 lentivirus. (B) RT-qPCR of BZW1 expression (left), cell proliferation assay (middle), and LDAs (right) in 83 and X01 GSCs infected with shCtrl and shCD97 lentivirus, followed by BZW1 lentivirus infection. (C–F) IB analysis of p-S6K, p-AKT, S6K, AKT, ARHGAP1, BZW1, and BZW2 levels in three different GSCs (83, X01, and 528 cells) treated with Torin1 48 h (C), AKT inhibitor IV 24 h (D), 24 h rapamycin (E), and JR-AB2-011 24 h (F). (G) Cell proliferation assays (left) and LDAs (right) were performed on 83 and X01 GSCs after treatment with JR-AB2-011. (H) Kaplan-Meier survival curves of mice orthotopically implanted with X01-Luc cells ( n = 5, 1 × 10 5 cells/mouse) and intraperitoneally (i.p.) treated with JR-AB2-011 (4 mg/kg) or vehicle. MST, median survival time. Log rank (Mantel-Cox) test. (I) Schematic representation of the CD97-related signaling pathway regulating the proliferation, self-renewal, and tumor progression of GSCs. Vinculin and GAPDH, and β-actin were used as loading controls in IB, β-actin was used as a loading control in RT-PCR. All error bars represent mean ± SD ( n = 3 independent experiments) in (B) and (G). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗ p < 0.05, t test. See also Figures S12–S15 .

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Inhibition, Infection, Quantitative RT-PCR, Expressing, Proliferation Assay, Control, Reverse Transcription Polymerase Chain Reaction

Journal: Cell Reports Medicine

Article Title: CD97 maintains tumorigenicity of glioblastoma stem cells via mTORC2 signaling and is targeted by CAR Th9 cells

doi: 10.1016/j.xcrm.2024.101844

Figure Lengend Snippet:

Article Snippet: containing Torin1 , SelleckChem , Cat# S2827.

Techniques: Produced, Virus, Plasmid Preparation, Recombinant, Purification, Cell Culture, Cell Isolation, Reporter Gene Assay, cDNA Synthesis, Apoptosis Assay, Cytotoxicity Assay, Gene Expression, shRNA, Sequencing, Amplification, Software, Microscopy, Western Blot

Journal: The Science of the Total Environment

Article Title: Sampling methods and assays applied in SARS-CoV-2 exposure assessment

doi: 10.1016/j.scitotenv.2021.145903

Figure Lengend Snippet: Data obtained from the chosen articles.

Article Snippet: , 43. Aerosol and surface contamination of SARS-CoV-2 observed in quarantine and isolation care , USA , No , Surface and air samples from COVID-19 patient rooms , Air sampling: Sartorius Airport MD8 air sampler operating at 50 Lpm for 15 min. Surface samples: sterile swabs , Viral RNA Extractions: using a Qiagen DSP Virus Spin Kit. RT-qPCR: using Invitrogen Superscript III Platinum One-Step Quantitative RT-qPCR System. Primers and probe used target the E gene of SARS-CoV-2. , • We detected viral contamination among all samples. , ( ) .

Techniques: Sampling, Lysis, RNA Extraction, Environmental Monitoring, Virus, Multiplex Assay, Northern Blot, Marker, RNA Detection, Isolation, Membrane, Control, Environmental Sampling, Amplification, Transmission Assay, Aerosol, Diagnostic Assay, Infection, Sterility, Real-time Polymerase Chain Reaction, Nested PCR, Reverse Transcription, Extraction, Purification, Digital PCR, Preserving, Quantitative RT-PCR, cDNA Synthesis, Magnetic Beads, Incubation, Modification, One Step RT-PCR, Cell Culture, Sequencing

Journal: Journal of cellular physiology

Article Title: Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation

doi: 10.1002/jcp.25399

Figure Lengend Snippet: GCs synergize with Runx2 in stimulating a select gene set. (A) ST2/Rx2dox mesenchymal pluripotent cells were treated for 48 h in quadruplicate with vehicle control (C), dox (to induce Runx2; D), the synthetic GC dex (1 μM; G), or dox and dex together (DG). Global gene expression was profiled using the Illumina BeadChip platform and the response of genes to dex in the presence of dox is illustrated as a volcano plot, with red and blue symbols representing genes that were stimulated or repressed, respectively, by dox alone (D/C ≥ 2 or D/C ≤ −2, respectively; FDR P < 0.05). Data representing genes that did not significantly respond to dox alone by these criteria are in gray. NC, no change. Triangles represent genes that were strongly stimulated by dex in the absence of dox (G/C ≥ 5, FDR P < 0.05). Dashed lines highlight a set of 8 genes at the top right that were strongly stimulated (≥ 8 fold; P < 1e–08) by dex in the presence of dox. (B–G) ST2/Rx2dox cells were treated for 24, 48, or 72 h with dox and/or dex at the depicted concentrations, and expression of the indicated genes was measured by RT-qPCR. Data are means and SDs (n = 3) relative to the control values at 24 h, defined as 1. [Color figure can be viewed at wileyonlinelibrary.com].

Article Snippet: Quantitative RT-qPCR Between 0.7 and 1 μg RNA, extracted as above, was reverse-transcribed using qScript™ cDNA SuperMix synthesis kit (Quanta BioSciences, Inc., Gaithersburg, MD).

Techniques: Expressing, Quantitative RT-PCR

Journal: Journal of cellular physiology

Article Title: Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation

doi: 10.1002/jcp.25399

Figure Lengend Snippet: Synergistic stimulation of Wif1 by Runx2 and GCs in primary osteoblast cultures. Pre-osteoblasts isolated from newborn mouse calvariae were transiently transfected with RUNX2 or a control plasmid and then treated for 72 h with 1.0 μM dex or vehicle. Expression of the indicated genes was assessed by either Western blot analysis of whole cell extracts (A) or (B–E). (F, G) Untransfected cells were treated with differentiation medium on day 2 of culture. Dex (1 μM) or vehicle was added for 72 h before lysis, followed by Western blot analysis of Runx2 (F) and RT-qPCR analysis of Wif1 (G) on days 5 and 7. Bars represent mean ± SD (n = 3).

Article Snippet: Quantitative RT-qPCR Between 0.7 and 1 μg RNA, extracted as above, was reverse-transcribed using qScript™ cDNA SuperMix synthesis kit (Quanta BioSciences, Inc., Gaithersburg, MD).

Techniques: Isolation, Transfection, Plasmid Preparation, Expressing, Western Blot, Lysis, Quantitative RT-PCR

Journal: Journal of cellular physiology

Article Title: Glucocorticoids Hijack Runx2 to Stimulate Wif1 for Suppression of Osteoblast Growth and Differentiation

doi: 10.1002/jcp.25399

Figure Lengend Snippet: Effect of Wif1 silencing on Wnt target gene expression in ST2 cells expressing Runx2 and treated with GCs. A–B. ST2/Rx2dox cells were transduced with lentiviruses encoding a nonspecific hairpin RNA (shNS) or either of two hairpins (shWif11 or shWif12) targeting distinct regioion in Wif1 mRNA. The derived shRNA-expressing sub-lines were treated for 72 h with dox and/or dex as indicated and Wif1 expression was measured by RT-qPCR (A, Mean ± SD; n = 3) and by Western blotting (B). +/− signs indicate presence or absence of dox and dex in both A and B. The RNA data in A are corrected for 18S RNA and Coomassie blue-stained proteins that remained in the SDS–PAGE gels after transfer are shown as a loading control in B. (C, D) The RNAs from Figure 4A were subjected to RT-qPCR analysis of Axin2 and Ccnd1 (mean ± SD, n = 3).

Article Snippet: Quantitative RT-qPCR Between 0.7 and 1 μg RNA, extracted as above, was reverse-transcribed using qScript™ cDNA SuperMix synthesis kit (Quanta BioSciences, Inc., Gaithersburg, MD).

Techniques: Expressing, Transduction, Derivative Assay, shRNA, Quantitative RT-PCR, Western Blot, Staining, SDS Page